肿节风提取工艺及肿节风注射液中主要成分的研究
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摘要
肿节风为《中国药典》收载的法定药材。主要用于抗肿瘤、抗炎等。肿节风制剂临床疗效显著,但由于肿节风中异秦皮啶等有效成分含量低,制剂制备方法陈旧,质量标准低,影响疗效,因此对肿节风制剂改进制备工艺,提高质量,进行二次开发是十分必要的。肿节风药材的提取工艺及肿节风中指标性成分异秦皮啶的富集纯化技术是提高制剂质量的关键。
采用均匀设计、正交设计优选肿节风的提取工艺,确定提取工艺。使异秦皮啶在提取物中的含量达到5.7%,提取率88.5%。建立了肿节风提取物的指纹图谱,提取物的指纹图谱与市售肿节风注射液的基本重合。考察了提取工艺中异秦皮啶作为指标的合理性,首次提出“相对质量”的概念,应用“相对质量”对肿节风药材中未知组分的提取工艺进行了优选。用D101大孔树脂对肿节风粗提物中异秦皮啶进行富集纯化研究。确定了异秦皮啶富集的最佳工艺。富集的干膏中含异秦皮啶20.7%,最终提取率71.0%。
从肿节风注射液中分离出了7个化合物,鉴定出了2个,分别为β-谷甾醇和异秦皮啶,得到340mg可作为对照品的异秦皮啶。7个化合物及几种肿节风注射液中已知成分进行抑制基质金属蛋白酶活性的测定,发现其中一种成分有良好的抑制基质金属蛋白酶-9的活性。
Herba Sarcandrae is a kind of legal crude drugs that is recorded in " Chinese pharmacopoeia " .It is very extensive to use in antitumor treatment and resisting the inflammation, etc..
It is riper that the preparation of Herba Sarcandrae is used in curing inflammation, such as angina, etc. But there is no great breakthrough in antitumor research, as well as resisting rheumatism and rheumatoid, etc. Though the clinical curative effect of some Herba Sarcandrae preparations is remarkable, the preparation methods and controlling quality with the quality level decades ago have still been used mostly, and it’s reported that the bad reaction of Herba Sarcandrae preparation appeared in the clinical practice. So it is very essential to improve process of preparing, raise the quality and carry on the secondary development. It is key to enrich the effective position and improve extraction process of Herba Sarcandrae for improving the quality of Herba Sarcandrae preparation.
Isofraxidin is the index composition in Herba Sarcandrae, but a trifle of absolute content, account for 0.02%~0.1% of the dry complete grass, in addition the extraction craft lags behind, mostly adopting the method of extraction with water then precipitated with ethanol , so the content of the compositions , such as isofraxidin, etc. are very low in the preparation, only account for 10～340μg/ml,the result of treatment is difficult to guarantee, which has restricted the development and application further of Herba Sarcandrae and isofraxidin. So it is to be badly in need of study the craft of enrichment isofraxidin and extraction of Herba Sarcandrae. Isofraxidin, as the main active composition of Herba Sarcandrae, is the key index of Herba Sarcandrae extracting.
It may make the breakthrough achievement that preparations of precise curative effect , the backbone in the preparation, the function of the backbone and these respects of function mechanism are studied connectedly at the same time .
The extraction method of isofraxidin from Herba Sarcandrae has the solvent extraction, membrane separation etc. at present. The solvent extraction needs to use a large number of organic solvents, the cost high, unsafety, extraction cycle long; membrane separation with high costs and unsatisfactory result, isofraxidin is low in content, and apt to stop up the membrane hole,which results the membrane recycled difficultly.
This subject adopts uniform design, orthogonal design to make the extraction process of Herba Sarcandrae designing optimum, including the solvent kind , the solvent dosage, extraction time, extraction number of times etc.. We confirm that the optimal process was obtained as follows: The Herba Sarcandrae was refluxed three times with total 8 times of 95% EtOH, 1 h for each time, since isofraxidin content rises to 5.7% in extracts from 0.102% in the crude drugs, extraction percentage is 88.5%. We have set up HPLC fingerprint atlas of Herba Sarcandrae extracts, HPLC fingerprint atlas of extracts and HPLC fingerprint atlas of the Herba Sarcandrae injection that the market sells are coincident basically. We have investigated the rationality of isofraxidin as the index in the extraction process of Herba Sarcandrae and the influence of each extraction factor to other component parts content in Herba Sarcandrae extracts.
Because most traditional Chinese medicines contain many kinds of chemical compounds, the effective composition is indeterminate, one or several kinds of composition are often adopted as the index to choose extraction process or make quality criteria, other many kinds of composition are neglected, so it is difficult to really improve and controll quality of traditional Chinese medicinal materials and preparation. As seeking the optimum extraction process of traditional Chinese medicinal materials, extraction percentage of many kinds of component parts in traditional Chinese medicinal materials is improved more overall , through comparing " relative quality "of other main component parts which is gotten by the index composition in extracts. While determining the index composition of traditional Chinese medicinal materials with HPLC, adopting " relative quality " can control the content of other many kinds of composition in crude drugs, which controls the quality of the crude drugs , does not need to determine the content of every kind of composition in the crude drugs(that is impossible in fact), calculating " relative quality " and using HPLC table picture in determining the index composition of traditional Chinese medicinal materials, do not need to increase determining time and the cost. Compared with the method by fingerprint atlas controlling traditional Chinese medicinal materials quality, the law of " relative quality " needn't make peaks of every composition completely separate, and calculate with each peak group, which is easy to implement and popularize.
We select D101 macroporous resin which is used more at domestic and of which organic solvent residual control and fore treatment are more maturity and polyamide to enrich isofraxidin in crude extract of Herba Sarcandrae. By studying the dynamic course absorbed, saturation point, the ratios of the diameter of macroporous resin post with height, the flow velocity influence of the sample liquid on absorbing isofraxidin by the macroporous resin , the kinds of adsorption stripping liquid, the influence of the elutriant flow rate on isofraxidin adsorption stripping, the influence the sample amount on resin regeneration, the isofraxidin enrichment process from Herba Sarcandrae extractive is obtained as follows: Extractive is dissolved in the hot water, sample solution is prepared 0.5g(dried medicinal herb)/ml, the sample solution passes macroporous resin, the ratios of the diameter of macroporous resin post with height is 1:15, at adsorbtion stage sample solution passes macroporous resin with the flow rate of 2V/h, samples is unsaturation , sample amount is 0.8g(dried medicinal herb)/ml(macroporous resin); the volume of 40% EtOH as many as 5 times column volume is used to adsorp stripping, adsorption stripping rate is 2V/h, EtOH from adsorption stripping liquid is reclaimed, gained extract is dried.Dry extract have 20.7% isofraxidin, ultimum percentage from Herba Sarcandrae is 71.0%.
By these separation methods such as column chromatography, TLC, recrystallisation and so on 7 compounds from Herba Sarcandrae injection is gotten, two compounds is identified, one isβ-sitosterine and the other is isofraxidin; 340mg isofraxidin which can use as standard preparation is gotten.
The studies in recent years discover that matrix metalloproteinase (MMP) promotes the transformation of the tumour and the development of rheumatoid arthritis. There is antineoplastic regarding MMP as the target on the market already. Herba Sarcandrae injection is used for treating the tumour extensively, and is proved tentatively that it is not antitumor function of cell's toxicity to play, Herba Sarcandrae injection is used for treating the rheumatoid arthritis. These evidences indicate that compound as MMP inhibitor may be contained in Herba Sarcandrae.
So we determin the inhibiting MMP activity in 7 compounds which are spearated from Herba Sarcandrae injection and the others major compounds that exist in Herba Sarcandrae injection. A kind of compound found can inhibit MMP-9. The compound in Herba Sarcandrae maybe is the key component of antitumor and resisting rheumatoid arthritis, which maybe can developed as new antitumor drug.
采用均匀设计、正交设计优选肿节风的提取工艺,确定提取工艺。使异秦皮啶在提取物中的含量达到5.7%,提取率88.5%。建立了肿节风提取物的指纹图谱,提取物的指纹图谱与市售肿节风注射液的基本重合。考察了提取工艺中异秦皮啶作为指标的合理性,首次提出“相对质量”的概念,应用“相对质量”对肿节风药材中未知组分的提取工艺进行了优选。用D101大孔树脂对肿节风粗提物中异秦皮啶进行富集纯化研究。确定了异秦皮啶富集的最佳工艺。富集的干膏中含异秦皮啶20.7%,最终提取率71.0%。
从肿节风注射液中分离出了7个化合物,鉴定出了2个,分别为β-谷甾醇和异秦皮啶,得到340mg可作为对照品的异秦皮啶。7个化合物及几种肿节风注射液中已知成分进行抑制基质金属蛋白酶活性的测定,发现其中一种成分有良好的抑制基质金属蛋白酶-9的活性。
Herba Sarcandrae is a kind of legal crude drugs that is recorded in " Chinese pharmacopoeia " .It is very extensive to use in antitumor treatment and resisting the inflammation, etc..
It is riper that the preparation of Herba Sarcandrae is used in curing inflammation, such as angina, etc. But there is no great breakthrough in antitumor research, as well as resisting rheumatism and rheumatoid, etc. Though the clinical curative effect of some Herba Sarcandrae preparations is remarkable, the preparation methods and controlling quality with the quality level decades ago have still been used mostly, and it’s reported that the bad reaction of Herba Sarcandrae preparation appeared in the clinical practice. So it is very essential to improve process of preparing, raise the quality and carry on the secondary development. It is key to enrich the effective position and improve extraction process of Herba Sarcandrae for improving the quality of Herba Sarcandrae preparation.
Isofraxidin is the index composition in Herba Sarcandrae, but a trifle of absolute content, account for 0.02%~0.1% of the dry complete grass, in addition the extraction craft lags behind, mostly adopting the method of extraction with water then precipitated with ethanol , so the content of the compositions , such as isofraxidin, etc. are very low in the preparation, only account for 10～340μg/ml,the result of treatment is difficult to guarantee, which has restricted the development and application further of Herba Sarcandrae and isofraxidin. So it is to be badly in need of study the craft of enrichment isofraxidin and extraction of Herba Sarcandrae. Isofraxidin, as the main active composition of Herba Sarcandrae, is the key index of Herba Sarcandrae extracting.
It may make the breakthrough achievement that preparations of precise curative effect , the backbone in the preparation, the function of the backbone and these respects of function mechanism are studied connectedly at the same time .
The extraction method of isofraxidin from Herba Sarcandrae has the solvent extraction, membrane separation etc. at present. The solvent extraction needs to use a large number of organic solvents, the cost high, unsafety, extraction cycle long; membrane separation with high costs and unsatisfactory result, isofraxidin is low in content, and apt to stop up the membrane hole,which results the membrane recycled difficultly.
This subject adopts uniform design, orthogonal design to make the extraction process of Herba Sarcandrae designing optimum, including the solvent kind , the solvent dosage, extraction time, extraction number of times etc.. We confirm that the optimal process was obtained as follows: The Herba Sarcandrae was refluxed three times with total 8 times of 95% EtOH, 1 h for each time, since isofraxidin content rises to 5.7% in extracts from 0.102% in the crude drugs, extraction percentage is 88.5%. We have set up HPLC fingerprint atlas of Herba Sarcandrae extracts, HPLC fingerprint atlas of extracts and HPLC fingerprint atlas of the Herba Sarcandrae injection that the market sells are coincident basically. We have investigated the rationality of isofraxidin as the index in the extraction process of Herba Sarcandrae and the influence of each extraction factor to other component parts content in Herba Sarcandrae extracts.
Because most traditional Chinese medicines contain many kinds of chemical compounds, the effective composition is indeterminate, one or several kinds of composition are often adopted as the index to choose extraction process or make quality criteria, other many kinds of composition are neglected, so it is difficult to really improve and controll quality of traditional Chinese medicinal materials and preparation. As seeking the optimum extraction process of traditional Chinese medicinal materials, extraction percentage of many kinds of component parts in traditional Chinese medicinal materials is improved more overall , through comparing " relative quality "of other main component parts which is gotten by the index composition in extracts. While determining the index composition of traditional Chinese medicinal materials with HPLC, adopting " relative quality " can control the content of other many kinds of composition in crude drugs, which controls the quality of the crude drugs , does not need to determine the content of every kind of composition in the crude drugs(that is impossible in fact), calculating " relative quality " and using HPLC table picture in determining the index composition of traditional Chinese medicinal materials, do not need to increase determining time and the cost. Compared with the method by fingerprint atlas controlling traditional Chinese medicinal materials quality, the law of " relative quality " needn't make peaks of every composition completely separate, and calculate with each peak group, which is easy to implement and popularize.
We select D101 macroporous resin which is used more at domestic and of which organic solvent residual control and fore treatment are more maturity and polyamide to enrich isofraxidin in crude extract of Herba Sarcandrae. By studying the dynamic course absorbed, saturation point, the ratios of the diameter of macroporous resin post with height, the flow velocity influence of the sample liquid on absorbing isofraxidin by the macroporous resin , the kinds of adsorption stripping liquid, the influence of the elutriant flow rate on isofraxidin adsorption stripping, the influence the sample amount on resin regeneration, the isofraxidin enrichment process from Herba Sarcandrae extractive is obtained as follows: Extractive is dissolved in the hot water, sample solution is prepared 0.5g(dried medicinal herb)/ml, the sample solution passes macroporous resin, the ratios of the diameter of macroporous resin post with height is 1:15, at adsorbtion stage sample solution passes macroporous resin with the flow rate of 2V/h, samples is unsaturation , sample amount is 0.8g(dried medicinal herb)/ml(macroporous resin); the volume of 40% EtOH as many as 5 times column volume is used to adsorp stripping, adsorption stripping rate is 2V/h, EtOH from adsorption stripping liquid is reclaimed, gained extract is dried.Dry extract have 20.7% isofraxidin, ultimum percentage from Herba Sarcandrae is 71.0%.
By these separation methods such as column chromatography, TLC, recrystallisation and so on 7 compounds from Herba Sarcandrae injection is gotten, two compounds is identified, one isβ-sitosterine and the other is isofraxidin; 340mg isofraxidin which can use as standard preparation is gotten.
The studies in recent years discover that matrix metalloproteinase (MMP) promotes the transformation of the tumour and the development of rheumatoid arthritis. There is antineoplastic regarding MMP as the target on the market already. Herba Sarcandrae injection is used for treating the tumour extensively, and is proved tentatively that it is not antitumor function of cell's toxicity to play, Herba Sarcandrae injection is used for treating the rheumatoid arthritis. These evidences indicate that compound as MMP inhibitor may be contained in Herba Sarcandrae.
So we determin the inhibiting MMP activity in 7 compounds which are spearated from Herba Sarcandrae injection and the others major compounds that exist in Herba Sarcandrae injection. A kind of compound found can inhibit MMP-9. The compound in Herba Sarcandrae maybe is the key component of antitumor and resisting rheumatoid arthritis, which maybe can developed as new antitumor drug.
引文
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